The results summarized below were obtained in collaborative studies: To identify the mAChR subtype that mediates cholinergic vasodilation in murine retinal arterioles, retinal vascular preparations from mAChR-deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and non-muscarinic vasoactive drugs were measured by video microscopy. The outcome of these studies indicated that endothelial M3 mAChRs mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase. (Gericke A, et al. Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles. Invest. Ophthalmol. Vis. Sci. 52, 7479-84, 2011) To examine the potential role of M1 mAChRs in cognition, M1 receptor-deficient mice were tested using the five-choice serial reaction time test of attentional and response functions, carried out using a computer-automated touchscreen test system. The mice were also tested on several tasks featuring learning, memory and perceptual challenges. Surprisingly, M1 receptor deficiency had no effect on attentional function per se. However, the M1 receptor mutant mice showed impairments in object recognition memory and various other behavioral responses. (Bartko SJ, et al. Intact attentional processing but abnormal responding in M1 muscarinic receptor-deficient mice using an automated touchscreen method. Neuropharmacology 61, 1366-78, 2011) Recent studies suggest that enhanced signaling through mAChRs is a key feature of Fmr1 KO mice, a mouse model of fragile X syndrome (FXS). To examine whether enhanced M4 mAChR activity contributes to the behavioral deficits typical for these mutant mice, we generated and analyzed Fmr1 KO mice that contained only one functional M4 receptor allele. Interestingly, reduction of M4 receptor expression in these mice rescued some, but not all, of the behavioral deficits displayed by the Fmr1 KO mice. These findings indicated that enhanced M4 receptor activity contributes to certain behavioral phenotypes in this mouse model of FXS. (Veeraragavan S, et al. Genetic reduction of muscarinic M4 receptor modulates analgesic response and acoustic startle response in a mouse model of fragile X syndrome (FXS). Behav Brain Res 228, 1-8, 2012) Studies with M1/M4 receptor double-knockout mice suggested that M1 and/or M4 receptors mediate the ability of muscarinic agonists to attenuate cocaine discrimination and self-administration in mice. To further explore this concept, we carried out additional cocaine discrimination studies with M1 and M4 receptor single KO mice, using a series of new ligands including an ectopic M1 receptor agonist (VU0357017) and an M1 receptor-selective positive allosteric modulator (BQCA). The outcome of these experiments support the concept that M1 receptor-selective agonists and mixed M1/M4 agonists may prove useful for the treatment of cocaine addiction. (Thomsen M, et al. Contribution of both M1 and M4 receptors to muscarinic agonist-mediated attenuation of the cocaine discriminative stimulus in mice. Psychopharmacology (Berlin) 220, 673-85, 2012) The anterolateral cell group of the bed nucleus of the stria terminalis (BNST/ALG) serves as an important relay station in stress circuitry. Limbic inputs into the BNST/ALG are primarily glutamatergic and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Studies with M2 mAChR KO mice, together with receptor localization experiments, indicated that presynaptic M2 receptors mediate the long-lasting reduction of the amplitude of stimulus-evoked EPSCs observed with WT mice. These data suggest that presynaptic M2 receptors represent an important modulator of the stress circuit. (Guo JD, et al. Presynaptic muscarinic M2 receptors modulate glutamatergic transmission in the bed nucleus of the stria terminalis. Neuropharmacology 62, 1671-83, 2012) Recent studies suggest that ACh can be synthesized and released from airway epithelial cells. Using different experimental techniques, we demonstrated that non-neuronal ACh can activate transepithelial ion currents in airway epithelia. Studies with mAChR mutant mice revealed that both M1 and M3 receptors are involved in mediating this activity. This newly identified mAChR-dependent pathway may represent a novel target for the treatment of airway diseases. (Hollenhorst MI, et al. Luminal cholinergic signalling in airway lining fluid: a novel mechanism for activating chloride secretion via Ca2+ -dependent Cl- and K+ channels. Br J Pharmacol 166, 1388-402, 2012) Several studies have shown that mAChR signaling plays a critical role in hippocampus-dependent learning and memory. However, the role of specific mAChRs in modulating the plasticity at associational/commissural (A/C) fiber-CA3 synapses remains unclear. Studies with M2 receptor KO mice demonstrated that M2 receptors promoted short-term facilitation and long-term potentiation (LTP) at the A/C synapse. Surprisingly, M2 receptors mediated the opposite effect on LTP at the mossy fiber (MF) synapse. These findings indicate that cholinergic projections recruit M2 receptors to redistribute the gain of LTP in CA3 pyramidal cells in an input-specific manner. (Zheng F, et al. M2 muscarinic acetylcholine receptors regulate long-term potentiation at hippocampal CA3 pyramidal cell synapses in an input-specific fashion. J Neurophysiol 108, 91-100, 2012) Previous work has shown that central M4 receptors play a role in regulating dopamine release in striatal areas. To test the potential therapeutic relevance of this finding, we examined the effects of VU0152100, an M4 receptor-selective positive allosteric modulator, on cocaine-induced behavioral and neurochemical effects in mice. In WT mice, VU0152100 caused a prominent reduction in cocaine self-administration, cocaine-induced hyperlocomotion, and cocaine-induced striatal dopamine increase. Selective deletion of the M4 receptor gene in dopamine D1 receptor-expressing neurons resulted in an attenuation of these effects. These results support the concept that positive allosteric modulators of the M4 receptor could prove useful for the treatment of cocaine abuse. (Dencker D, et al. An allosteric enhancer of M4 muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine. Psychopharmacology (Berl) May 31, 2012, Epub ahead of print)